The invention relates to a process for detecting mutations in the DNA of a transgenic mammal or a transgenic mammalian cell, which DNA contains one or more copies of a marker gene located in a bacterial cloning vector, comprising isolating DNA from cells of the transgenic mammal or the transgenic mammalian cells, recovering the vector from the isolated DNA, transforming a suitable bacterial host with the recovered vector and determining mutations that have occurred in the marker gene on the basis of expression of the marker gene in the host.
The invention relates more particularly to a transgenic animal model provided with a marker gene which is integrated in the genome and which, after isolation of chromosomal DNA, can be recovered efficiently. After transfer of the marker gene to a bacteria incapable of host restriction, a selection is made on the presence of a mutated marker gene to determine the mutation frequency.
Such an animal model is of importance inter alia for testing agents for carcinogenic properties. All newly obtained compounds are in principle suspected of carcinogenicity. According to present day insights they could change the structure of heritable material present in every cell core. Such changes of the heritable material (mutations) can result among other things in cancer. Mutagenic agents (agents which cause mutations in the DNA) are as far as is known also carcinogenic and can likewise cause mutations in the germ cells, which can lead to hereditary genetic deviations. The mutagenicity of an agent is considered the most important criterion in determining the risk of developing cancer or hereditary deviations. It is therefore of particular importance to be able to determine the mutagenicity of an agent at an early stage.
The best known process to date for testing potentially mutagenic agents is the Ames test (Ames, 1973) wherein mutations are detected in bacteria. Although this test can be performed quickly because of the short generation times of a bacteria, extensive research has demonstrated that the predictive value of the Ames test is not 100% (Hay, 1988; Ashby and Tennant, 1988). Preference is therefore generally given to a combination of tests including tests with cultured (in vitro) mammalian cells.
There are at this moment only a few possibilities of detecting mutations in vivo in cells of higher animals. An example is the HPRT test, wherein mutations in the hypoxanthine phosphoribosyl transferase (HPRT) gene are detected on the basis of the 6-thioguanine resistance of the mutated cells (Albertini, 1982). This process is however labour-intensive and only allows measurements on cells that can still divide and are simple to culture. In addition, the artificial nature of the in vitro situation is a drawback to this process, whereby it is difficult to draw conclusions concerning induced mutations in organs and tissues and possible differences between them. An alternative hereto is the use of laboratory animals (particularly rodents) in long-term studies. Animals are exposed herein to a suspect agent and there is a wait to see whether or not tumours appear. This is a time-consuming process however which can last several years and requires many animals, particularly when low concentrations of a suspect agent are being tested. This makes the test very expensive, whereby agents which are positive in the Ames test are often not tested further and do not therefore become commercially available.
A good alternative is the use of transgenic animals as described by Vijg and Uitterlinden (1987), Lohman et al. (1987) and Gossen et al. (1989) and in the European patent application EP-A-0 353 812. The transgenic mouse model described therein contains in each body cell, including the germ cells, a bacteriophage lambda vector in which the bacterial lacZ gene is cloned which serves as the mutation target gene. The vector can be recovered in efficient manner from chromosomal DNA by means of in vitro packaging of the vector in "empty" phage particles. The vector-containing phages are subsequently plated with E. coli C host cells (which are host restriction-negative) wherein the selection takes place between mutated lacZ vectors (colourless) and non-mutated lacZ vectors (blue). The ratio between the number of colourless plaques and the number of blue plaques then indicates the mutation frequency. Rescue of marker genes from total chromosomal mammalian DNA in this efficient manner was first described by Gossen and Vijg (1988; see also the European patent application EP-A-0 353 812).
Using this process, mutation induction can in principle be studied in any tissue or organ of the above described transgenic animals. A drawback to this process however is that recovery of the bacteriophage lambda vectors by means of in vitro packaging is relatively expensive and labour-intensive. This is inherent to the nature of the vector which must be recovered and the principle forming the basis of determining the mutation frequency, namely the ratio between the number of mutated colourless plaques and the number of non-mutated blue plaques. With respect to the nature of the bacteriophage lambda vector which must be recovered, experiments have indicated that there exists a great degree of variability between different batches of packaging extracts; this means that prior to use of each separate batch the efficiency must first be determined. With respect to determining the mutation frequency on the basis of the ratio between the number of colourless mutated plaques and the number of blue non-mutated plaques, it can be stated that a reverse system wherein mutant vectors are coloured and non-mutant vectors are colourless is to be recommended. The spontaneous mutation frequency in different tissues and organs is in any case in the order of 1:100,000. In order to obtain reliable mutation frequencies in different tissues and organs at least 1 to 1.5 million plaques per organ or tissue have to be analyzed with the present system. The analysis of these numbers of plaques is exceptionally labour-intensive and requires large quantities of the packaging extract, the substrate X-gal and the petri dishes necessary for the analysis.
The invention now provides a process and an animal model which obviate these drawbacks.